Surface-bound, unimolecular, double-stranded DNA

ABSTRACT

Libraries of unimolecular, double-stranded oligonucleotides on a solid support. These libraries are useful in pharmaceutical discovery for the screening of numerous biological samples for specific interactions between the double-stranded oligonucleotides, and peptides, proteins, drugs and RNA. In a related aspect, the present invention provides libraries of conformationally restricted probes on a solid support. The probes are restricted in their movement and flexibility using double-stranded oligonucleotides as scaffolding. The probes are also useful in various screening procedures associated with drug discovery and diagnosis. The present invention further provides methods for the preparation and screening of the above libraries.

GOVERNMENT RIGHTS

Research leading to the invention was funded in part by NIH Grant No.______, and the government may have certain rights to the invention.

BACKGROUND OF THE INVENTION

The present invention relates to the field of polymer synthesis and theuse of polymer libraries for biological screening. More specifically, inone embodiment the invention provides arrays of diverse double-strandedoligonucleotide sequences. In another embodiment, the invention providesarrays of conformationally restricted probes, wherein the probes areheld in position using double-stranded DNA sequences as scaffolding.Libraries of diverse unimolecular double-stranded nucleic acid sequencesand probes may be used, for example, in screening studies fordetermination of binding affinity exhibited by binding proteins, drugs,or RNA.

Methods of synthesizing desired single stranded DNA sequences are wellknown to those of skill in the art. In particular, methods ofsynthesizing oligonucleotides are found in, for example, OligonucleotideSynthesis: A Practical Approach, Gait, ed., IRL Press, Oxford (1984),incorporated herein by reference in its entirety for all purposes.Synthesizing unimolecular double-stranded DNA in solution has also beendescribed. See, Durand, et al. Nucleic Acids Res. 18: 6353-6359 (1990)and Thomson, et al. Nucleic Acids Res. 21: 5600-5603 (1993), thedisclosures of both being incorporated herein by reference.

Solid phase synthesis of biological polymers has been evolving since theearly “Merrifield” solid phase peptide synthesis, described inMerrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963), incorporated hereinby reference for all purposes. Solid-phase synthesis techniques havebeen provided for the synthesis of several peptide sequences on, forexample, a number of “pins.” See e.g., Geysen et al., J. Immun. Meth.102: 259-274 (1987), incorporated herein by reference for all purposes.Other solid-phase techniques involve, for example, synthesis of variouspeptide sequences on different cellulose disks supported in a column.See Frank and Doring, Tetrahedron 44: 6031-6040 (1988), incorporatedherein by reference for all purposes. Still other solid-phase techniquesare described in U.S. Pat. No. 4,728,502 issued to Hamill and WO90/00626 (Beattie, inventor).

Each of the above techniques produces only a relatively low densityarray of polymers. For example, the technique described in Geysen et al.is limited to producing 96 different polymers on pins spaced in thedimensions of a standard microtiter plate.

Improved methods of forming large arrays of oligonucleotides, peptidesand other polymer sequences in a short period of time have been devised.Of particular note, Pirrung et al., U.S. Pat. No. 5,143,854 (see alsoPCT Application No. WO 90/15070) and Fodor et al., PCT Publication No.WO 92/10092, all incorporated herein by reference, disclose methods offorming vast arrays of peptides, oligonucleotides and other polymersequences using, for example, light-directed synthesis techniques. Seealso, Fodor et al., Science, 251: 767-777 (1991), also incorporatedherein by reference for all purposes. These procedures are now referredto as VLSIPS™ procedures.

In the above-referenced Fodor et al., PCT application, an elegant methodis described for using a computer-controlled system to direct a VLSIPS™procedure. Using this approach, one heterogenous array of polymers isconverted, through simultaneous coupling at a number of reaction sites,into a different heterogenous array. See, U.S. application Ser. Nos.07/796,243 and 07/980,523, the disclosures of which are incorporatedherein for all purposes.

The development of VLSIPS™ technology as described in the above-notedU.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and92/10092, is considered pioneering technology in the fields ofcombinatorial synthesis and screening of combinatorial libraries. Morerecently, patent application Ser. No. 08/082,937, filed Jun. 25, 1993,describes methods for making arrays of oligonucleotide probes that canbe used to check or determine a partial or complete sequence of a targetnucleic acid and to detect the presence of a nucleic acid containing aspecific oligonucleotide sequence.

A number of biochemical processes of pharmaceutical interest involve theinteraction of some species, e.g., a drug, a peptide or protein, or RNA,with double-stranded DNA. For example, protein/DNA binding interactionsare involved with a number of transcription factors as well as tumorsuppression associated with the p53 protein and the genes contributingto a number of cancer conditions.

SUMMARY OF THE INVENTION

High-density arrays of diverse unimolecular, double-strandedoligonucleotides, as well as arrays of conformationally restrictedprobes and methods for their use are provided by virtue of the presentinvention. In addition, methods and devices for detecting duplexformation of oligonucleotides on an array of diverse single-strandedoligonucleotides are also provided by this invention. Further, anadhesive based on the specific binding characteristics of two arrays ofcomplementary oligonucleotides is provided in the present invention.

According to one aspect of the present invention, libraries ofunimolecular, double-stranded oligonucleotides are provided. Each memberof the library is comprised of a solid support, an optional spacer forattaching the double-stranded oligonucleotide to the support and forproviding sufficient space between the double-stranded oligonucleotideand the solid support for subsequent binding studies and assays, anoligonucleotide attached to the spacer and further attached to a secondcomplementary oligonucleotide by means of a flexible linker, such thatthe two oligonucleotide portions exist in a double-strandedconfiguration. More particularly, the members of the libraries of thepresent invention can be represented by the formula:Y-L¹-X¹-L²-X²in which Y is a solid support, L¹ is a bond or a spacer, L² is aflexible linking group, and X¹ and X² are a pair of complementaryoligonucleotides.

In a specific aspect of the invention, the library of differentunimolecular, double-stranded oligonucleotides can be used for screeninga sample for a species which binds to one or more members of thelibrary.

In a related aspect of the invention, a library of differentconformationally-restricted probes attached to a solid support isprovided. The individual members each have the formula:—X¹¹-Z-X¹²in which X¹¹ and X¹² are complementary oligonucleotides and Z is a probehaving sufficient length such that X¹¹ and X¹² form a double-strandedoligonucleotide portion of the member and thereby restrict theconformations available to the probe. In a specific aspect of theinvention, the library of different conformationally-restricted probescan be used for screening a sample for a species which binds to one ormore probes in the library.

According to vet another aspect of the present invention, methods anddevices for the bioelectronic detection of duplex formation areprovided.

According to still another aspect of the invention, an adhesive isprovided which comprises two surfaces of complementary oligonucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1F illustrate the preparation of a member of a library ofsurface-bound, unimolecular double-stranded DNA as well as bindingstudies with receptors having specificity for either the double strandedDNA portion, a probe which is held in a conformationally restricted formby DNA scaffolding, or a bulge or loop region of RNA.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Abbreviations

The following abbreviations are used herein: phi, phenanthrenequinonediimine; phen′, 5-amido-glutaric acid-1,10-phenanthroline; dppz,dipyridophenazine.

Glossary

The following terms are intended to have the following general meaningsas they are used herein:

Chemical terms: As used herein, the term “alkyl” refers to a saturatedhydrocarbon radical which may be straight-chain or branched-chain (forexample, ethyl, isopropyl, t-amyl, or 2,5-dimethylhexyl). When “alkyl”or “alkylene” is used to refer to a linking group or a spacer, it istaken to be a group having two available valences for covalentattachment, for example, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH(CH₃)CH₂— and—H₂(CH₂CH₂)₂CH₂—. Preferred alkyl groups as substituents are thosecontaining 1 to 10 carbon atoms, with those containing 1 to 6 carbonatoms being particularly preferred. Preferred alkyl or alkylene groupsas linking groups are those containing 1 to 20 carbon atoms, with thosecontaining 3 to 6 carbon atoms being particularly preferred. The term“polyethylene glycol” is used to refer to those molecules which haverepeating units of ethylene glycol, for example, hexaethylene glycol(HO—(CH₂CH₂O)₅—H₂CH₂OH). When the term “polyethylene glycol” is used torefer to linking groups and spacer groups, it would be understood by oneof skill in the art that other polyethers or polyols could be used aswell (i.e, polypropylene glycol or mixtures of ethylene and propyleneglycols).

The term “protecting group” as used herein, refers to any of the groupswhich are designed to block one reactive site in a molecule while achemical reaction is carried out at another reactive site. Moreparticularly, the protecting groups used herein can be any of thosegroups described in Greene, et al., Protective Groups In OrganicChemistry, 2nd Ed., John Wiley & Sons, New York, N.Y., 1991,incorporated herein by reference. The proper selection of protectinggroups for a particular synthesis will be governed by the overallmethods employed in the synthesis. For example, in “light-directed”synthesis, discussed below, the protecting groups will be photolabileprotecting groups such as NVOC, MeNPOC, and those disclosed inco-pending Application PCT/US93/10162 (filed Oct. 22, 1993),incorporated herein by reference. In other methods, protecting groupsmay be removed by chemical methods and include groups such as FMOC, DMTand others known to those of skill in the art.

Complementary or substantially complementary: Refers to thehybridization or base pairing between nucleotides or nucleic acids, suchas, for instance, between the two strands of a double stranded DNAmolecule or between an oligonucleotide primer and a primer binding siteon a single stranded nucleic acid to be sequenced or amplified.Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andcompared and with appropriate nucleotide insertions or deletions, pairwith at least about 80% of the nucleotides of the other strand, usuallyat least about 90% to 95%, and more preferably from about 98 to 100%.

Alternatively, substantial complementarity exists when an RNA or DNAstrand will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementarity over a stretch of at least 14 to 25nucleotides, preferably at least about 75%, more preferably at leastabout 90% complementarity. See, M. Kanehisa Nucleic Acids Res. 12: 203(1984), incorporated herein by reference.

Stringent hybridization conditions will typically include saltconcentrations of less than about 1 M, more usually less than about 500mM and preferably less than about 200 mM. Hybridization temperatures canbe as low as 5° C., but are typically greater than 22° C. more typicallygreater than about 30° C. and preferably in excess of about 37° C.Longer fragments may require higher hybridization temperatures forspecific hybridization. As other factors may affect the stringency ofhybridization, including base composition and length of thecomplementary strands, presence of organic solvents and extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one alone.

Epitope: The portion of an antigen molecule which is delineated by thearea of interaction with the subclass of receptors known as antibodies.

Identifier tag: A means whereby one can identify which molecules haveexperienced a particular reaction in the synthesis of an oligomer. Theidentifier tag also records the step in the synthesis series in whichthe molecules experienced that particular monomer reaction. Theidentifier tag may be any recognizable feature which is, for example:microscopically distinguishable in shape, size, color, optical density,etc.; differently absorbing or emitting of light; chemically reactive;magnetically or electronically encoded; or in some other waydistinctively marked with the required information. A preferred exampleof such an identifier tag is an oligonucleotide sequence.

Ligand/Probe: A ligand is a molecule that is recognized by a particularreceptor. The agent bound by or reacting with a receptor is called a“ligand,” a term which is definitionally meaningful only in terms of itscounterpart receptor. The term “ligand” does not imply any particularmolecular size or other structural or compositional feature other thanthat the substance in question is capable of binding or otherwiseinteracting with the receptor. Also, a ligand may serve either as thenatural ligand to which the receptor binds, or as a functional analoguethat may act as an agonist or antagonist. Examples of ligands that canbe investigated by this invention include, but are not restricted to,agonists and antagonists for cell membrane receptors, toxins and venoms,viral epitopes, hormones (e.g., opiates, steroids, etc.), hormonereceptors, peptides, enzymes, enzyme substrates, substrate analogs,transition state analogs, cofactors, drugs, proteins, and antibodies.The term “probe” refers to those molecules which are expected to actlike ligands but for which binding information is typically unknown. Forexample, if a receptor is known to bind a ligand which is a peptideβ-turn, a “probe” or library of probes will be those molecules designedto mimic the peptide β-turn. In instances where the particular ligandassociated with a given receptor is unknown, the term probe refers tothose molecules designed as potential ligands for the receptor.

Monomer: Any member of the set of molecules which can be joined togetherto form an oligomer or polymer. The set of monomers useful in thepresent invention includes, but is not restricted to, for the example ofoligonucleotide synthesis, the set of nucleotides consisting of adenine,thymine, cytosine, guanine, and uridine (A, T, C, G, and U,respectively) and synthetic analogs thereof. As used herein, monomersrefers to any member of a basis set for synthesis of an oligomer.Different basis sets of monomers may be used at successive steps in thesynthesis of a polymer.

Oligomer or Polymer: The oligomer or polymer sequences of the presentinvention are formed from the chemical or enzymatic addition of monomersubunits. Such oligomers include, for example, both linear, cyclic, andbranched polymers of nucleic acids, polysaccharides, phospholipids, andpeptides having either α-, β-, or ω-amino acids, heteropolymers in whicha known drug is covalently bound to any of the above, polyurethanes,polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines,polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or otherpolymers which will be readily apparent to one skilled in the art uponreview of this disclosure. As used herein, the term oligomer or polymeris meant to include such molecules as β-turn mimetics, prostaglandinsand benzodiazepines which can also be synthesized in a stepwise fashionon a solid support.

Peptide: A peptide is an oligomer in which the monomers are amino acidsand which are joined together through amide bonds and alternativelyreferred to as a polypeptide. In the context of this specification itshould be appreciated that when α-amino acids are used, they may be theL-optical isomer or the D-optical isomer. Other amino acids which areuseful in the present invention include unnatural amino acids such asβ-alanine, phenylglycine, homoarginine and the like. Peptides are morethan two amino acid monomers long, and often more than 20 amino acidmonomers long. Standard abbreviations for amino acids are used (e.g., Pfor proline). These abbreviations are included in Stryer, Biochemistry,Third Ed. (1988), which is incorporated herein by reference for allpurposes.

Oligonucleotides: An oligonucleotide is a single-stranded DNA or RNAmolecule, typically prepared by synthetic means. Alternatively,naturally occurring oligonucleotides, or fragments thereof, may beisolated from their natural sources or purchased from commercialsources. Those oligonucleotides employed in the present invention willbe 4 to 100 nucleotides in length, preferably from 6 to 30 nucleotides,although oligonucleotides of different length may be appropriate.Suitable oligonucleotides may be prepared by the phosphoramidite methoddescribed by Beaucage and Carruthers, Tetrahedron Lett., 22: 1859-1862(1981), or by the triester method according to Matteucci, et al., J. Am.Chem. Soc. 103: 3185 (1981), both incorporated herein by reference, orby other chemical methods using either a commercial automatedoligonucleotide synthesizer or VLSIPS™ technology (discussed in detailbelow). When oligonucleotides are referred to as “double-stranded.” itis understood by those of skill in the art that a pair ofoligonucleotides exist in a hydrogen-bonded, helical array typicallyassociated with, for example, DNA. In addition to the 100% complementaryform of double-stranded oligonucleotides, the term “double-stranded” asused herein is also meant to refer to those forms which include suchstructural features as bulges and loops, described more fully in suchbiochemistry texts as Stryer, Biochemistry, Third Ed., (1988),previously incorporated herein by reference for all purposes.

Receptor: A molecule that has an affinity for a given ligand or probe.Receptors may be naturally-occurring or manmade molecules. Also, theycan be employed in their unaltered natural or isolated state or asaggregates with other species. Receptors may be attached, covalently ornoncovalently, to a binding member, either directly or via a specificbinding substance. Examples of receptors which can be employed by thisinvention include, but are not restricted to, antibodies, cell membranereceptors, monoclonal antibodies and antisera reactive with specificantigenic determinants (such as on viruses, cells or other materials),drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins,sugars, polysaccharides, cells, cellular membranes, and organelles.Receptors are sometimes referred to in the art as anti-ligands. As theterm receptors is used herein, no difference in meaning is intended. A“ligand-receptor pair” is formed when two molecules have combinedthrough molecular recognition to form a complex.

Other examples of receptors which can be investigated by this inventioninclude but are not restricted to:

-   -   a) Microorganism receptors: Determination of ligands or probes        that bind to receptors, such as specific transport proteins or        enzymes essential to survival of microorganisms, is useful in a        new class of antibiotics. Of particular value would be        antibiotics against opportunistic fungi, protozoa, and those        bacteria resistant to the antibiotics in current use.    -   b) Enzymes: For instance, the binding site of enzymes such as        the enzymes responsible for cleaving neurotransmitters.        Determination of ligands or probes that bind to certain        receptors, and thus modulate the action of the enzymes that        cleave the different neurotransmitters, is useful in the        development of drugs that can be used in the treatment of        disorders of neurotransmission.    -   c) Antibodies: For instance, the invention may be useful in        investigating the ligand-binding site on the antibody molecule        which combines with the epitope of an antigen of interest.        Determining a sequence that mimics an antigenic epitope may lead        to the development of vaccines of which the immunogen is based        on one or more of such sequences, or lead to the development of        related diagnostic agents or compounds useful in therapeutic        treatments such as for autoimmune diseases (e.g., by blocking        the binding of the “self” antibodies).    -   d) Nucleic Acids: The invention may be useful in investigating        sequences of nucleic acids acting as binding sites for cellular        proteins (“trans-acting factors”). Such sequences may include,        e.g., transcription factors, suppressors, enhancers or promoter        sequences.    -   e) Catalytic Polypeptides: Polymers, preferably polypeptides,        which are capable of promoting a chemical reaction involving the        conversion of one or more reactants to one or more products.        Such polypeptides generally include a binding site specific for        at least one reactant or reaction intermediate and an active        functionality proximate to the binding site, which functionality        is capable of chemically modifying the bound reactant. Catalytic        polypeptides are described in. Lerner, R. A. et al. Science 252:        659 (1991), which is incorporated herein by reference.    -   f) Hormone receptors: For instance, the receptors for insulin        and growth hormone. Determination of the ligands which bind with        high affinity to a receptor is useful in the development of, for        example, an oral replacement of the daily injections which        diabetics must take to relieve the symptoms of diabetes, and in        the other case, a replacement for the scarce human growth        hormone that can only be obtained from cadavers or by        recombinant DNA technology. Other examples are the        vasoconstrictive hormone receptors; determination of those        ligands that bind to a receptor may lead to the development of        drugs to control blood pressure.    -   g) Opiate receptors: Determination of ligands that bind to the        opiate receptors in the brain is useful in the development of        less-addictive replacements for morphine and related drugs.

Substrate or Solid Support: A material having a rigid or semi-rigidsurface. Such materials will preferably take the form of plates orslides, small beads, pellets, disks or other convenient forms, althoughother forms may be used. In some embodiments, at least one surface ofthe substrate will be substantially flat. In other embodiments, aroughly spherical shape is preferred.

Synthetic: Produced by in vitro chemical or enzymatic synthesis. Thesynthetic libraries of the present invention may be contrasted withthose in viral or plasmid vectors, for instance, which may be propagatedin bacterial, yeast, or other living hosts.

DESCRIPTION OF THE INVENTION

The broad concept of the present invention is illustrated in FIG. 1.FIGS. 1A, 1B and 1C illustrate the preparation of surface-boundunimolecular double stranded DNA, while FIGS. 1D, 1E, and 1F illustrateuses for the libraries of the present invention.

FIG. 1A shows a solid support 1 having an attached spacer 2, which isoptional. Attached to the distal end of the spacer is a first oligomer3, which can be attached as a single unit or synthesized on the supportor spacer in a monomer by monomer approach. FIG. 1B shows a subsequentstage in the preparation of one member of a library according to thepresent invention. In this stage, a flexible linker 4 is attached to thedistal end of the oligomer 3. In other embodiments, the flexible linkerwill be a probe. FIG. 1C shows the completed surface-bound unimoleculardouble stranded DNA which is one member of a library, wherein a secondoligomer 5 is now attached to the distal end of the flexible linker (orprobe). As shown in FIG. 1C, the length of the flexible linker (orprobe) 4 is sufficient such that the first and second oligomers (whichare complementary) exist in a double-stranded conformation. It will beappreciated by one of skill in the art, that the libraries of thepresent invention will contain multiple, individually synthesizedmembers which can be screened for various types of activity. Three suchbinding events are illustrated in FIGS. 1D, 1E and 1F.

In FIG. 1D, a receptor 6, which can be a protein, RNA molecule or othermolecule which is known to bind to DNA, is introduced to the library.Determining which member of a library binds to the receptor providesinformation which is useful for diagnosing diseases, sequencing DNA orRNA, identifying genetic characteristics, or in drug discovery.

In FIG. 1E, the linker 4 is a probe for which binding information issought. The probe is held in a conformationally restricted manner by theflanking oligomers 3 and 5, which are present in a double-strandedconformation. As a result, a library of conformationally restrictedprobes can be screened for binding activity with a receptor 7 which hasspecificity for the probe.

The present invention also contemplates the preparation of libraries ofunimolecular, double-stranded oligonucleotides having bulges or loops inone of the strands as depicted in FIG. 1F. In FIG. 1F, oneoligonucleotide 5 is shown as having a bulge 8. Specific RNA bulges areoften recognized by proteins (e.g., TAR RNA is recognized by the TATprotein of HIV). Accordingly, libraries of RNA bulges or loops areuseful in a number of diagnostic applications. One of skill in the artwill appreciate that the bulge or loop can be present in eitheroligonucleotide portion 3 or 5.

Libraries of Unimolecular, Double-Stranded Oligonucleotides

In one aspect, the present invention provides libraries of unimoleculardouble-stranded oligonucleotides, each member of the library having theformula:Y-L¹-X¹-L²-X²in which Y represents a solid support. X¹ and X² represent a pair ofcomplementary oligonucleotides. L¹ represents a bond or a spacer, and L²represents a linking group having sufficient length such that X¹ and X²form a double-stranded oligonucleotide.

The solid support may be biological, nonbiological, organic, inorganic,or a combination of any of these, existing as particles, strands,precipitates, gels, sheets, tubing, spheres, containers, capillaries,pads, slices, films, plates, slides, etc. The solid support ispreferably flat but may take on alternative surface configurations. Forexample, the solid support may contain raised or depressed regions onwhich synthesis takes place. In some embodiments, the solid support willbe chosen to provide appropriate light-absorbing characteristics. Forexample, the support may be a polymerized Langmuir Blodgett film,functionalized glass. Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene,polycarbonate, or combinations thereof. Other suitable solid supportmaterials will be readily apparent to those of skill in the art.Preferably, the surface of the solid support will contain reactivegroups, which could be carboxyl, amino, hydroxyl, thiol, or the like.More preferably, the surface will be optically transparent and will havesurface Si—OH functionalities, such as are found on silica surfaces.

Attached to the solid support is an optional spacer, L¹. The spacermolecules are preferably of sufficient length to permit thedouble-stranded oligonucleotides in the completed member of the libraryto interact freely with molecules exposed to the library. The spacermolecules, when present, are typically 6-50 atoms long to providesufficient exposure for the attached double-stranded DNA molecule. Thespacer, L¹, is comprised of a surface attaching portion and a longerchain portion. The surface attaching portion is that part of L¹ which isdirectly attached to the solid support. This portion can be attached tothe solid support via carbon-carbon bonds using, for example, supportshaving (poly)trifluorochloroethylene surfaces, or preferably, bysiloxane bonds (using, for example, glass or silicon oxide as the solidsupport). Siloxane bonds with the surface of the support are formed inone embodiment via reactions of surface attaching portions bearingtrichlorosilyl or trialkoxysilyl groups. The surface attaching groupswill also have a site for attachment of the longer chain portion. Forexample, groups which are suitable for attachment to a longer chainportion would include amines, hydroxyl, thiol, and carboxyl. Preferredsurface attaching portions include aminoalkylsilanes andhydroxyalkylsilanes. In particularly preferred embodiments, the surfaceattaching portion of L¹ is eitherbis(2-hydroxyethyl)-aminopropyltriethoxysilane.2-hydroxyethylaminopropyltriethoxysilane, aminopropyltriethoxysilane orhydroxypropyltriethoxysilane.

The longer chain portion can be any of a variety of molecules which areinert to the subsequent conditions for polymer synthesis. These longerchain portions will typically be aryl acetylene, ethylene glycololigomers containing 2-14 monomer units, diamines, diacids, amino acids,peptides, or combinations thereof. In some embodiments, the longer chainportion is a polynucleotide. The longer chain portion which is to beused as part of L¹ can be selected based upon itshydrophilic/hydrophobic properties to improve presentation of thedouble-stranded oligonucleotides to certain receptors, proteins ordrugs. The longer chain portion of L¹ can be constructed ofpolyethyleneglycols, polynucleotides, alkylene, polyalcohol, polyester,polyamine, polyphosphodiester and combinations thereof. Additionally,for use in synthesis of the libraries of the invention, L¹ willtypically have a protecting group, attached to a functional group (i.e.,hydroxyl, amino or carboxylic acid) on the distal or terminal end of thechain portion (opposite the solid support). After deprotection andcoupling, the distal end is covalently bound to an oligomer.

Attached to the distal end of L¹ is an oligonucleotide, X¹, which is asingle-stranded DNA or RNA molecule. The oligonucleotides which are partof the present invention are typically of from about 4 to about 100nucleotides in length. Preferably, X¹ is an oligonucleotide which isabout 6 to about 30 nucleotides in length. The oligonucleotide istypically linked to L¹ via the 3′-hydroxyl group of the oligonucleotideand a functional group on L¹ which results in the formation of an ether,ester, carbamate or phosphate ester linkage.

Attached to the distal end of X¹ is a linking group, L², which isflexible and of sufficient length that X¹ can effectively hybridize withX². The length of the linker will typically be a length which is atleast the length spanned by two nucleotide monomers, and preferably atleast four nucleotide monomers, while not be so long as to interferewith either the pairing of X¹ and X² or any subsequent assays. Thelinking group itself will typically be an alkylene group (of from about6 to about 24 carbons in length), a polyethyleneglycol group (of fromabout 2 to about 24 ethyleneglycol monomers in a linear configuration),a polyalcohol group, a polyamine group (e.g., spermine, spermidine andpolymeric derivatives thereof), a polyester group (e.g., poly(ethylacrylate) having of from 3 to 15 ethyl acrylate monomers in a linearconfiguration), a polyphosphodiester group, or a polynucleotide (havingfrom about 2 to about 12 nucleic acids). Preferably, the linking groupwill be a polyethyleneglycol group which is at least atetraethyleneglycol, and more preferably, from about 1 to 4hexaethyleneglycols linked in a linear array. For use in synthesis ofthe compounds of the invention, the linking group will be provided withfunctional groups which can be suitably protected or activated. Thelinking group will be covalently attached to each of the complementaryoligonucleotides, X¹ and X², by means of an ether, ester, carbamate,phosphate ester or amine linkage. The flexible linking group L² will beattached to the 5′-hydroxyl of the terminal monomer of X¹ and to the3′-hydroxyl of the initial monomer of X². Preferred linkages arephosphate ester linkages which can be formed in the same manner as theoligonucleotide linkages which are present in X¹ and X². For example,hexaethyleneglycol can be protected on one terminus with a photolabileprotecting group (i.e., NVOC or MeNPOC) and activated on the otherterminus with 2-cyanoethyl-N,N-diisopropylamino-chlorophosphite to forma phosphoramidite. This linking group can then be used for constructionof the libraries in the same manner as the photolabile-protected,phosphoramidite-activated nucleotides. Alternatively, ester linkages toX¹ and X² can be formed when the L² has terminal carboxylic acidmoieties (using the 5′-hydroxyl of X¹ and the 3′-hydroxyl of X²). Othermethods of forming ether, carbamate or amine linkages are known to thoseof skill in the art and particular reagents and references can be foundin such texts as March, Advanced Organic Chemistry, 4th Ed.,Wiley-Interscience, New York, N.Y., 1992, sincorporated herein byreference.

The oligonucleotide, X², which is covalently attached to the distal endof the linking group is, like X¹, a single-stranded DNA or RNA molecule.The oligonucleotides which are part of the present invention aretypically of from about 4 to about 100 nucleotides in length.Preferably, X² is an oligonucleotide which is about 6 to about 30nucleotides in length and exhibits complementarity to X¹ of from 90 to100%. More preferably, X¹ and X² are 100% complementary. In one group ofembodiments, either X¹ or X² will further comprise a bulge or loopportion and exhibit complementarity of from 90 to 100% over theremainder of the oligonucleotide.

In a particularly preferred embodiment, the solid support is a silicasupport, the spacer is a polyethyleneglycol conjugated to anaminoalkylsilane, the linking group is a polyethyleneglycol group, andX¹ and X² are complementary oligonucleotides each comprising of from 6to 30 nucleic acid monomers.

The library can have virtually any number of different members, and willbe limited only by the number or variety of compounds desired to bescreened in a given application and by the synthetic capabilities of thepractitioner. In one group of embodiments, the library will have from 2up to 100 members. In other groups of embodiments, the library will havebetween 100 and 10000 members, and between 10000 and 1000000 members,preferably on a solid support. In preferred embodiments, the librarywill have a density of more than 100 members at known locations percm⁻¹, preferably more than 1000 per cm², more preferably more than10,000 per cm².

Libraries of Conformationally Restricted Probes

In still another aspect, the present invention provides libraries ofconformationally-restricted probes. Each of the members of the librarycomprises a solid support having an optional spacer, which is attachedto an oligomer of the formula:-X¹¹-Z-X¹²in which X¹¹ and X¹² are complementary oligonucleotides and Z is aprobe. The probe will have sufficient length such that X¹¹ and X¹² forma double-stranded DNA portion of each member. X¹¹ and X¹² are asdescribed above for X¹ and X² respectively, except that for the presentaspect of the invention, each member of the probe library can have thesame X¹¹ and the same X¹², and differ only in the probe portion. In onegroup of embodiments, X¹¹ and X¹² are either a poly-A oligonucleotide ora poly-T oligonucleotide.

As noted above, each member of the library will typically have adifferent probe portion. The probes, Z, can be any of a variety ofstructures for which receptor-probe binding information is sought forconformationally-restricted forms. For example, the probe can be anagonist or antagonist for a cell membrane receptor, a toxin, venom,viral epitope, hormone, peptide, enzyme, cofactor, drug, protein orantibody. In one group of embodiments, the probes are differentpeptides, each having of from about 4 to about 12 amino acids.Preferably the probes will be linked via polyphosphate diesters,although other linkages are also suitable. For example, the last monomeremployed on the X¹¹ chain can be a 5′-aminopropyl-functionalizedphosphoramidite nucleotide (available from Glen Research. Sterling, Va.USA or Genosys Biotechnologies, The Woodlands. Texas. USA) which willprovide a synthesis initiation site for the carboxy to amino synthesisof the peptide probe. Once the peptide probe is formed, a3′-succinylated nucleoside (from Cruachem, Sterling, Va. USA) will beadded under peptide coupling conditions. In yet another group ofembodiments, the probes will be oligonucleotides of from 4 to about 30nucleic acid monomers which will form a DNA or RNA hairpin structure.For use in synthesis, the probes can also have associated functionalgroups (i.e., hydroxyl, amino, carboxylic acid, anhydride andderivatives thereof) for attaching two positions on the probe to each ofthe complementary oligonucleotides.

The surface of the solid support is preferably provided with a spacermolecule, although it will be understood that the spacer molecules arenot elements of this aspect of the invention. Where present, the spacermolecules will be as described above for L¹.

The libraries of conformationally restricted probes can also havevirtually any number of members. As above, the number of members will belimited only by design of the particular screening assay for which thelibrary will be used, and by the synthetic capabilities of thepractitioner. In one group of embodiments, the library will have from 2to 100 members. In other groups of embodiments, the library will havebetween 100 and 10000 members, and between 10000 and 1000000 members.Also as above, in preferred embodiments, the library will have a densityof more than 100 members at known locations per cm², preferably morethan 1000 per cm², more preferably more than 10,000 per cm².

Preparation of the Libraries

The present invention further provides methods for the preparation ofdiverse unimolecular, double-stranded oligonucleotides on a solidsupport. In one group of embodiments, the surface of a solid support hasa plurality of preselected regions. An oligonucleotide of from 6 to 30monomers is formed on each of the preselected regions. A linking groupis then attached to the distal end of each of the oligonucleotides.Finally, a second oligonucleotide is formed on the distal end of eachlinking group such that the second oligonucleotide is complementary tothe oligonucleotide already present in the same preselected region. Thelinking group used will have sufficient length such that thecomplementary oligonucleotides form a unimolecular, double-strandedoligonucleotide. In another group of embodiments, each chemicallydistinct member of the library will be synthesized on a separate solidsupport.

Libraries on a Single Substrate

Light-Directed Methods

For those embodiments using a single solid support, the oligonucleotidesof the present invention can be formed using a variety of techniquesknown to those skilled in the art of polymer synthesis on solidsupports. For example. “light directed” methods (which are one techniquein a family of methods known as VLSIPS™ methods) are described in U.S.Pat. No. 5,143,854, previously incorporated by reference. The lightdirected methods discussed in the '854 patent involve activatingpredefined regions of a substrate or solid support and then contactingthe substrate with a preselected monomer solution. The predefinedregions can be activated with a light source, typically shown through amask (much in the manner of photolithography techniques used inintegrated circuit fabrication). Other regions of the substrate remaininactive because they are blocked by the mask from illumination andremain chemically protected. Thus, a light pattern defines which regionsof the substrate react with a given monomer. By repeatedly activatingdifferent sets of predefined regions and contacting different monomersolutions with the substrate, a diverse array of polymers is produced onthe substrate. Of course, other steps such as washing unreacted monomersolution from the substrate can be used as necessary. Other techniquesinclude mechanical techniques such as those described in PCT No.92/10183, U.S. Ser. No. 07/796,243, also incorporated herein byreference for all purposes. Still further techniques include bead basedtechniques such as those described in PCT US/93/04145, also incorporatedherein by reference, and pin based methods such as those described inU.S. Pat. No. 5,288,514, also incorporated herein by reference.

The VLSIPS™ methods are preferred for making the compounds and librariesof the present invention. The surface of a solid support, optionallymodified with spacers having photolabile protecting groups such as NVOCand MeNPOC, is illuminated through a photolithographic mask, yieldingreactive groups (typically hydroxyl groups) in the illuminated regions.A 3′-O-phosphoramidite activated deoxynucleoside (protected at the5′-hydroxyl with a photolabile protecting group) is then presented tothe surface and chemical coupling occurs at sites that were exposed tolight. Following capping, and oxidation, the substrate is rinsed and thesurface illuminated through a second mask, to expose additional hydroxylgroups for coupling. A second 5′-protected, 3′-O-phosphoramiditeactivated deoxynucleoside is presented to the surface. The selectivephotodeprotection and coupling cycles are repeated until the desired setof oligonucleotides is produced. Alternatively, an oligomer of from, forexample, 4 to 30 nucleotides can be added to each of the preselectedregions rather than synthesize each member in a monomer by monomerapproach. At this point in the synthesis, either a flexible linkinggroup or a probe can be attached in a similar manner. For example, aflexible linking group such as polyethylene glycol will typically havingan activating group (i.e., a phosphoramidite) on one end and aphotolabile protecting group attached to the other end. Suitablyderivatized polyethylene glycol linking groups can be prepared by themethods described in Durand, et al. Nucleic Acids Res. 18: 6353-6359(1990). Briefly, a polyethylene glycol (i.e., hexaethylene glycol) canbe mono-protected using MeNPOC-chloride. Following purification of themono-protected glycol, the remaining hydroxy moiety can be activatedwith 2-cyanoethyl-N,N-diisopropylamino-chlorophosphite. Once theflexible linking group has been attached to the first oligonucleotide(X¹), deprotection and coupling cycles will proceed using 5′-protected,3′-O-phosphoramidite activated deoxynucleosides or intact oligomers.Probes can be attached in a manner similar to that used for the flexiblelinking group. When the desired probe is itself an oligomer, it can beformed either in stepwise fashion on the immobilized oligonucleotide orit can be separately synthesized and coupled to the immobilized oligomerin a single step. For example, preparation of conformationallyrestricted β-turn mimetics will typically involve synthesis of anoligonucleotide as described above, in which the last nucleoside monomerwill be derivatized with an aminoalkyl-functionalized phosphoramidite.See, U.S. Pat. No. 5,288,514, previously incorporated by reference. Thedesired peptide probe is typically formed in the direction from carboxylto amine terminus. Subsequent coupling of a 3′-succinylated nucleoside,for example, provides the first monomer in the construction of thecomplementary oligonucleotide strand (which is carried out by the abovemethods). Alternatively, a library of probes can be prepared by firstderivatizing a solid support with multiple poly(A) or poly(T)oligonucleotides which are suitably protected with photolabileprotecting groups, deprotecting at known sites and constructing theprobe at those sites, then coupling the complementary poly(T) or poly(A)oligonucleotide.

Flow Channel or Spotting Methods

Additional methods applicable to library synthesis on a single substrateare described in co-pending application Ser. No. 07/980,523, filed Nov.20, 1992, and Ser. No. 07/796,243, filed Nov. 22, 1991, incorporatedherein by reference for all purposes. In the methods disclosed in theseapplications, reagents are delivered to the substrate by either (1)flowing within a channel defined on predefined regions or (2) “spotting”on predefined regions. However, other approaches, as well ascombinations of spotting and flowing, may be employed. In each instance,certain activated regions of the substrate are mechanically separatedfrom other regions when the monomer solutions are delivered to thevarious reaction sites.

A typical “flow channel” method applied to the compounds and librariesof the present invention can generally be described as follows. Diversepolymer sequences are synthesized at selected regions of a substrate orsolid support by forming flow channels on a surface of the substratethrough which appropriate reagents flow or in which appropriate reagentsare placed. For example, assume a monomer “A” is to be bound to thesubstrate in a first group of selected regions. If necessary, all orpart of the surface of the substrate in all or a part of the selectedregions is activated for binding by, for example, flowing appropriatereagents through all or some of the channels, or by washing the entiresubstrate with appropriate reagents. After placement of a channel blockon the surface of the substrate, a reagent having the monomer A flowsthrough or is placed in all or some of the channel(s). The channelsprovide fluid contact to the first selected regions, thereby binding themonomer A on the substrate directly or indirectly (via a spacer) in thefirst selected regions.

Thereafter, a monomer B is coupled to second selected regions, some ofwhich may be included among the first selected regions. The secondselected regions will be in fluid contact with a second flow channel(s)through translation, rotation, or replacement of the channel block onthe surface of the substrate: through opening or closing a selectedvalve: or through deposition of a layer of chemical or photoresist. Ifnecessary, a step is performed for activating at least the secondregions. Thereafter, the monomer B is flowed through or placed in thesecond flow channel(s), binding monomer B at the second selectedlocations. In this particular example, the resulting sequences bound tothe substrate at this stage of processing will be, for example. A, B,and AB. The process is repeated to form a vast array of sequences ofdesired length at known locations on the substrate.

After the substrate is activated, monomer A can be flowed through someof the channels, monomer B can be flowed through other channels, amonomer C can be flowed through still other channels, etc. In thismanner, many or all of the reaction regions are reacted with a monomerbefore the channel block must be moved or the substrate must be washedand/or reactivated. By making use of many or all of the availablereaction regions simultaneously, the number of washing and activationsteps can be minimized.

One of skill in the art will recognize that there are alternativemethods of forming channels or otherwise protecting a portion of thesurface of the substrate. For example, according to some embodiments, aprotective coating such as a hydrophilic or hydrophobic coating(depending upon the nature of the solvent) is utilized over portions ofthe substrate to be protected, sometimes in combination with materialsthat facilitate wetting by the reactant solution in other regions. Inthis manner, the flowing solutions are further prevented from passingoutside of their designated flow paths.

The “spotting” methods of preparing compounds and libraries of thepresent invention can be implemented in much the same manner as the flowchannel methods. For example, a monomer A can be delivered to andcoupled with a first group of reaction regions which have beenappropriately activated. Thereafter, a monomer B can be delivered to andreacted with a second group of activated reaction regions. Unlike theflow channel embodiments described above, reactants are delivered bydirectly depositing (rather than flowing) relatively small quantities ofthem in selected regions. In some steps, of course, the entire substratesurface can be sprayed or otherwise coated with a solution. In preferredembodiments, a dispenser moves from region to region, depositing only asmuch monomer as necessary at each stop. Typical dispensers include amicropipette to deliver the monomer solution to the substrate and arobotic system to control the position of the micropipette with respectto the substrate, or an ink-jet printer. In other embodiments, thedispenser includes a series of tubes, a manifold, an array of pipettes,or the like so that various reagents can be delivered to the reactionregions simultaneously.

Pin-Based Methods

Another method which is useful for the preparation of compounds andlibraries of the present invention involves “pin based synthesis.” Thismethod is described in detail in U.S. Pat. No. 5,288,514, previouslyincorporated herein by reference. The method utilizes a substrate havinga plurality of pins or other extensions. The pins are each insertedsimultaneously into individual reagent containers in a tray. In a commonembodiment, an array of 96 pins/containers is utilized.

Each tray is filled with a particular reagent for coupling in aparticular chemical reaction on an individual pin. Accordingly, thetrays will often contain different reagents. Since the chemistrydisclosed herein has been established such that a relatively similar setof reaction conditions may be utilized to perform each of the reactions,it becomes possible to conduct multiple chemical coupling stepssimultaneously. In the first step of the process the invention providesfor the use of substrate(s) on which the chemical coupling steps areconducted. The substrate is optionally provided with a spacer havingactive sites. In the particular case of oligonucleotides, for example,the spacer may be selected from a wide variety of molecules which can beused in organic environments associated with synthesis as well asaqueous environments associated with binding studies. Examples ofsuitable spacers are polyethyleneglycols, dicarboxylic acids, polyaminesand alkylenes, substituted with, for example, methoxy and ethoxy groups.Additionally, the spacers will have an active site on the distal end.The active sites are optionally protected initially by protectinggroups. Among a wide variety of protecting groups which are useful areFMOC, BOC, t-butyl esters, t-butyl ethers, and the like. Variousexemplary protecting groups are described in, for example, Atherton etal., Solid Phase Peptide Synthesis, IRL Press (1989), incorporatedherein by reference. In some embodiments, the spacer may provide for acleavable function by way of, for example, exposure to acid or base.

Libraries on Multiple Substrates

Bead Based Methods

Yet another method which is useful for synthesis of compounds andlibraries of the present invention involves “bead based synthesis.” Ageneral approach for bead based synthesis is described copendingapplication Ser. No. 07/762,522 (filed Sep. 18, 1991); Ser. No.07/946,239 (filed Sep. 16, 1992); Ser. No. 08/146,886 (filed Nov. 2,1993); Ser. No. 07/876,792 (filed Apr. 29, 1992) and PCT/US93/04145(filed Apr. 28, 1993), the disclosures of which are incorporated hereinby reference.

For the synthesis of molecules such as oligonucleotides on beads, alarge plurality of beads are suspended in a suitable carrier (such aswater) in a container. The beads are provided with optional spacermolecules having an active site. The active site is protected by anoptional protecting group.

In a first step of the synthesis, the beads are divided for couplinginto a plurality of containers. For the purposes of this briefdescription, the number of containers will be limited to three, and themonomers denoted as A, B, C, D, E, and F. The protecting groups are thenremoved and a first portion of the molecule to be synthesized is addedto each of the three containers (i.e., A is added to container 1, B isadded to container 2 and C is added to container 3).

Thereafter, the various beads are appropriately washed of excessreagents, and remixed in one container. Again, it will be recognizedthat by virtue of the large number of beads utilized at the outset,there will similarly be a large number of beads randomly dispersed inthe container, each having a particular first portion of the monomer tobe synthesized on a surface thereof.

Thereafter, the various beads are again divided for coupling in anothergroup of three containers. The beads in the first container aredeprotected and exposed to a second monomer (D), while the beads in thesecond and third containers are coupled to molecule portions E and Frespectively. Accordingly, molecules AD, BD, and CD will be present inthe first container, while AE, BE, and CE will be present in the secondcontainer, and molecules AF, BF, and CF will be present in the thirdcontainer. Each bead, however, will have only a single type of moleculeon its surface. Thus, all of the possible molecules formed from thefirst portions A, B, C, and the second portions D, E, and F have beenformed.

The beads are then recombined into one container and additional stepssuch as are conducted to complete the synthesis of the polymermolecules. In a preferred embodiment, the beads are tagged with anidentifying tag which is unique to the particular double-strandedoligonucleotide or probe which is present on each bead. A completedescription of identifier tags for use in synthetic libraries isprovided in co-pending application Ser. No. 08/146,886 (filed Nov. 2,1993) previously incorporated by reference for all purposes.

Methods of Library Screening

A library prepared according to any of the methods described above canbe used to screen for receptors having high affinity for eitherunimolecular, double-stranded oligonucleotides or conformationallyrestricted probes. In one group of embodiments, a solution containing amarked (labelled) receptor is introduced to the library and incubatedfor a suitable period of time. The library is then washed free ofunbound receptor and the probes or double-stranded oligonucleotideshaving high affinity for the receptor are identified by identifyingthose regions on the surface of the library where markers are located.Suitable markers include, but are not limited to, radiolabels,chromophores, fluorophores, chemiluminescent moieties, and transitionmetals. Alternatively, the presence of receptors may be detected using avariety of other techniques, such as an assay with a labelled enzyme,antibody, and the like. Other techniques using various marker systemsfor detecting bound receptor will be readily apparent to those skilledin the art.

In a preferred embodiment, a library prepared on a single solid support(using, for example, the VLSIPS™ technique) can be exposed to a solutioncontaining marked receptor such as a marked antibody. The receptor canbe marked in any of a variety of ways, but in one embodiment marking iseffected with a radioactive label. The marked antibody binds with highaffinity to an immobilized antigen previously localized on the surface.After washing the surface free of unbound receptor, the surface isplaced proximate to x-ray film or phosphorimagers to identify theantigens that are recognized by the antibody. Alternatively, afluorescent marker may be provided and detection may be by way of acharge-coupled device (CCD), fluorescence microscopy or laser scanning.

When autoradiography is the detection method used, the marker is aradioactive label, such as ³²P. The marker on the surface is exposed toX-ray film or a phosphorimager, which is developed and read out on ascanner. An exposure time of about 1 hour is typical in one embodiment.Fluorescence detection using a fluorophore label, such as fluorescein,attached to the receptor will usually require shorter exposure times.

Quantitative assays for receptor concentrations can also be performedaccording to the present invention. In a direct assay method, thesurface containing localized probes prepared as described above, isincubated with a solution containing a marked receptor for a suitableperiod of time. The surface is then washed free of unbound receptor. Theamount of marker present at predefined regions of the surface is thenmeasured and can be related to the amount of receptor in solution.Methods and conditions for performing such assays are well-known and arepresented in, for example, L. Hood et al., Immunology, Benjamin/Cummings(1978), and E. Harlow et al., Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory, (1988). See, also U.S. Pat. No. 4,376,110 formethods of performing sandwich assays. The precise conditions forperforming these steps will be apparent to one skilled in the art.

A competitive assay method for two receptors can also be employed usingthe present invention. Methods of conducting competitive assays areknown to those of skill in the art. One such method involvesimmobilizing conformationally restricted probes on predefined regions ofa surface as described above. An unmarked first receptor is then boundto the probes on the surface having a known specific binding affinityfor the receptors. A solution containing a marked second receptor isthen introduced to the surface and incubated for a suitable time. Thesurface is then washed free of unbound reagents and the amount of markerremaining on the surface is measured. In another form of competitionassay, marked and unmarked receptors can be exposed to the surfacesimultaneously. The amount of marker remaining on predefined regions ofthe surface can be related to the amount of unknown receptor insolution. Yet another form of competition assay will utilize tworeceptors having different labels, for example, two differentchromophores.

In other embodiments, in order to detect receptor binding, thedouble-stranded oligonucleotides which are formed with attached probesor with a flexible linking group will be treated with an intercalatingdye, preferably a fluorescent dye. The library can be scanned toestablish a background fluorescence. After exposure of the library to areceptor solution, the exposed library will be scanned or illuminatedand examined for those areas in which fluorescence has changed.Alternatively, the receptor of interest can be labeled with afluorescent dye by methods known to those of skill in the art andincubated with the library of probes. The library can then be scanned orilluminated, as above, and examined for areas of fluorescence.

In instances where the libraries are synthesized on beads in a number ofcontainers, the beads are exposed to a receptor of interest. In apreferred embodiment the receptor is fluorescently or radioactivelylabelled. Thereafter, one or more beads are identified that exhibitsignificant levels of, for example, fluorescence using one of a varietyof techniques. For example, in one embodiment, mechanical separationunder a microscope is utilized. The identity of the molecule on thesurface of such separated beads is then identified using, for example,NMR, mass spectrometry, PCR amplification and sequencing of theassociated DNA, or the like. In another embodiment, automated sorting(i.e., fluorescence activated cell sorting) can be used to separatebeads (bearing probes) which bind to receptors from those which do notbind. Typically the beads will be labeled and identified by methodsdisclosed in Needels, et al., Proc. Natl. Acad. Sci., USA 90:10700-10704 (1993), incorporated herein by reference.

The assay methods described above for the libraries of the presentinvention will have tremendous application in such endeavors as DNA“footprinting” of proteins which bind DNA. Currently, DNA footprintingis conducted using DNase I digestion of double-stranded DNA in thepresence of a putative DNA binding protein. Gel analysis of cut andprotected DNA fragments then provides a “footprint” of where the proteincontacts the DNA. This method is both labor and time intensive. See,Galas et al., Nucleic Acid Res. 5: 3157 (1978). Using the above methods,a “footprint” could be produced using a single array of unimolecular,double-stranded oligonucleotides in a fraction of the time ofconventional methods. Typically, the protein will be labeled with aradioactive or fluorescent species and incubated with a library ofunimolecular, double-stranded DNA. Phosphorimaging or fluorescencedetection will provide a footprint of those regions on the library wherethe protein has bound. Alternatively, unlabeled protein can be used.When unlabeled protein is used, the double-stranded oligonucleotides inthe library will all be labeled with a marker, typically a fluorescentmarker. Incorporation of a marker into each member of the library can becarried out by terminating the oligonucleotide synthesis with acommercially available fluorescing phosphoramidite nucleotidederivative. Following incubation with the unlabeled protein, the librarywill be treated with DNase I and examined for areas which are protectedfrom cleavage.

The assay methods described above for the libraries of the presentinvention can also be used in reverse drug discovery. In such anapplication, a compound having known pharmacological safety or otherdesired properties (e.g., aspirin) could be screened against a varietyof double-stranded oligonucleotides for potential binding. If thecompound is shown to bind to a sequence associated with, for example,tumor suppression, the compound can be further examined for efficacy inthe related diseases.

In other embodiments, probe arrays comprising β-turn mimetics can beprepared and assayed for activity against a particular receptor. β-turnmimetics are compounds having molecular structures similar to β-turnswhich are one of the three major components in protein moleculararchitecture. β-turns are similar in concept to hairpin turns ofoligonucleotide strands, and are often critical recognition features forvarious protein-ligand and protein-protein interactions. As a result, alibrary of β-turn mimetic probes can provide or suggest new therapeuticagents having a particular affinity for a receptor which will correspondto the affinity exhibited by the β-turn and its receptor.

Bioelectronic Devices and Methods

In another aspect, the present invention provides a method for thebioelectronic detection of sequence-specific oligonucleotidehybridization. A general method and device which is useful indiagnostics in which a biochemical species is attached to the surface ofa sensor is described in U.S. Pat. No. 4,562,157 (the Lowe patent),incorporated herein by reference. The present method utilizes arrays ofimmobilized oligonucleotides (prepared, for example, using VLSIPS™technology) and the known photo-induced electron transfer which ismediated by a DNA double helix structure. See, Murphy et al., Science262: 1025-1029 (1993). This method is useful in hybridization-baseddiagnostics, as a replacement for fluorescence-based detection systems.The method of bioelectronic detection also offers higher resolution andpotentially higher sensitivity than earlier diagnostic methods involvingsequencing/detecting by hybridization. As a result, this method findsapplications in genetic mutation screening and primary sequencing ofoligonucleotides. The method can also be used for Sequencing ByHybridization (SBH), which is described in co-pending application Ser.No. 08/082,937 (filed Jun. 25, 1993) and Ser. No. 08/168,904 (filed Dec.15, 1993), each of which are incorporated herein by reference for allpurposes. This method uses a set of short oligonucleotide probes ofdefined sequence to search for complementary sequences on a longertarget strand of DNA. The hybridization pattern is used to reconstructthe target DNA sequence. Thus, the hybridization analysis of largenumbers of probes can be used to sequence long stretches of DNA. Inimmediate applications of this hybridization methodology, a small numberof probes can be used to interrogate local DNA sequence.

In the present inventive method, hybridization is monitored usingbioelectronic detection. In this method, the target DNA, or firstoligonucleotide, is provided with an electron-donor tag and thenincubated with an array of oligonucleotide probes, each of which bearsan electron-acceptor tag and occupies a known position on the surface ofthe array. After hybridization of the first oligonucleotide to the arrayhas occurred, the hybridized array is illuminated to induce an electrontransfer reaction in the direction of the surface of the array. Theelectron transfer reaction is then detected at the location on thesurface where hybridization has taken place. Typically, each of theoligonucleotide probes in an array will have an attachedelectron-acceptor tag located near the surface of the solid support usedin preparation of the array. In embodiments in which the arrays areprepared by light-directed methods (i.e, typically 3′ to 5′ direction),the electron-acceptor tag will be located near the 3′ position. Theelectron-acceptor tag can be attached either to the 3′ monomer bymethods known to those of skill in the art, or it can be attached to aspacing group between the 3′ monomer and the solid support. Such aspacing group will have, in addition to functional groups for attachmentto the solid support and the oligonucleotide, a third functional groupfor attachment of the electron-acceptor tag. The target oligonucleotidewill typically have the electron-donor tag attached at the 3′ position.Alternatively, the target oligonucleotide can be incubated with thearray in the absence of an electron-donor tag. Following incubation, theelectron-donor tag can be added in solution. The electron-donor tag willthen intercalate into those regions where hybridization has occurred. Anelectron transfer reaction can then be detected in those regions havinga continuous DNA double helix.

The electron-donor tag can be any of a variety of complexes whichparticipate in electron transfer reactions and which can be attached toan oligonucleotide by a means which does not interfere with the electrontransfer reaction. In preferred embodiments, the electron-donor tag is aruthenium (II) complex, more preferably a ruthenium (II) (phen′)₂(dppz)complex.

The electron-acceptor tag can be any species which, with theelectron-donor tag, will participate in an electron transfer reaction.An example of an electron-acceptor tag is a rhodium (III) complex. Apreferred electron-acceptor tag is a rhodium (III) (phi)₂(phen′)complex.

In a particularly preferred embodiment, the electron-donor tag is aruthenium (II) (phen′)₂(dppz) complex and the electron-acceptor tag is arhodium (III) (phi)₂(phen′) complex.

In still another aspect, the present invention provides a device for thebioelectronic detection of sequence-specific oligonucleotidehybridization. The device will typically consist of a sensor having asurface to which an array of oligonucleotides are attached. Theoligonucleotides will be attached in pre-defined areas on the surface ofthe sensor and have an electron-acceptor tag attached to eacholigonucleotide. The electron-acceptor tag will be a tag which iscapable of producing an electron transfer signal upon illumination of ahybridized species, when the complementary oligonucleotide bears anelectron-donating tag. The signal will be in the direction of the sensorsurface and be detected by the sensor.

In a preferred embodiment, the sensor surface will be a silicon-basedsurface which can sense the electronic signal induced and, if necessary,amplify the signal. The metal contacts on which the probes will besynthesized can be treated with an oxygen plasma prior to synthesis ofthe probes to enhance the silane adhesion and concentration on thesurface. The surface will further comprise a multi-gated field effecttransistor, with each gate serving as a sensor and differentoligonucleotides attached to each gate. The oligonucleotides willtypically be attached to the metal contacts on the sensor surface bymeans of a spacer group.

The spacer group should not be too long, in order to ensure that thesensing function of the device is easily activated by the bindinginteraction and subsequent illumination of the “tagged” hybridizedoligonucleotides. Preferably, the spacer group is from 3 to 12 atoms inlength and will be as described above for the surface modifying portionof the spacer group, L¹.

The oligonucleotides which are attached to the spacer group can beformed by any of the solid phase techniques which are known to those ofskill in the art. Preferably, the oligonucleotides are formed one baseat a time in the direction of the 3′ terminus to the 5′ terminus by the“light-directed” methods described above. The oligonucleotide can thenbe modified at the 3′ end to attach the electron-acceptor tag. A numberof suitable methods of attachment are known. For example, modificationwith the reagent Aminolink2 (from Applied Biosystems, Inc.) provides aterminal phosphate moiety which is derivatized with an aminohexylphosphate ester. Coupling of a carboxylic acid, which is present on theelectron-acceptor tag, to the amine can then be carried out using HOBTand DCC. Alternatively, synthesis of the oligonucleotide can begin witha suitably derivatized and protected monomer which can then bedeprotected and coupled to the electron-acceptor tag once the completeoligonucleotide has been synthesized.

The silica surface can also be replaced by silicon nitride oroxynitride, or by an oxide of another metal, especially aluminum,titanium (IV) or iron (III). The surface can also be any other film,membrane, insulator or semiconductor overlying the sensor which will notinterfere with the detection of electron transfer detection and to whichan oligonucleotide can be coupled.

Additionally, detection devices other than an FET can be used. Forexample, sensors such as bipolar transistors, MOS transistors and thelike are also useful for the detection of electron transfer signals.

Adhesives

In still another aspect, the present invention provides an adhesivecomprising a pair of surfaces, each having a plurality of attachedoligonucleotides, wherein the single-stranded oligonucleotides on onesurface are complementary to the single-stranded oligonucleotides on theother surface. The strength and position/orientation specificity can becontrolled using a number of factors including the number and length ofoligonucleotides on each surface, the degree of complementarity, and thespatial arrangement of complementary oligonucleotides on the surface.For example, increasing the number and length of the oligonucleotides oneach surface will provide a stronger adhesive. Suitable lengths ofoligonucleotides are typically from about 10 to about 70 nucleotides.Additionally, the surfaces of oligonucleotides can be prepared such thatadhesion occurs in an extremely position-specific manner by a suitablearrangement of complementary oligonucleotides in a specific pattern.Small deviations from the optimum spatial arrangement are energeticallyunfavorable as many hybridization bonds must be broken and are notreformed in any other relative orientation.

The adhesives of the present invention will find use in numerousapplications. Generally, the adhesives are useful for adhering twosurfaces to one another. More specifically, the adhesives will findapplication where biological compatibility of the adhesive is desired.An example of a biological application involves use in surgicalprocedures where tissues must be held in fixed positions during orfollowing the procedure. In this application, the surfaces of theadhesive will typically be membranes which are compatible with thetissues to which they are attached.

A particular advantage of the adhesives of the present invention is thatwhen they are formed in an orientation specific manner, the adhesiveportions will be “self-finding,” that is the system will go to thethermodynamic equilibrium in which the two sides are matched in thepredetermined, orientation specific manner.

EXAMPLES Example 1

This example illustrates the general synthesis of an array ofunimolecular, double-stranded oligonucleotides on a solid support.

Unimolecular double stranded DNA molecules were synthesized on a solidsupport using standard light-directed methods (VLSIPS™ protocols). Twohexaethylene glycol (PEG) linkers were used to covalently attach thesynthesized oligonucleotides to the derivatized glass surface. Synthesisof the first (inner) strand proceeded one nucleotide at a time usingrepeated cycles of photo-deprotection and chemical coupling of protectednucleotides. The nucleotides each had a protecting group on the baseportion of the monomer as well as a photolabile MeNPoc protecting groupon the 5′ hydroxyl. Upon completion of the inner strand, anotherMeNPoc-protected PEG linker was covalently attached to the 5′ end of thesurface-bound oligonucleotide. After addition of the internal PEGlinker, the PEG is photodeprotected, and the synthesis of the secondstrand proceeded in the normal fashion. Following the synthesis cycles,the DNA bases were deprotected using standard protocols. The sequence ofthe second (outer) strand, being complementary to that of the innerstrand, provided molecules with short, hydrogen bonded, unimoleculardouble-stranded structure as a result of the presence of the internalflexible PEG linker.

An array of 16 different molecules were synthesized on a derivatizedglass slide in order to determine whether short, unimolecular DNAstructures could be formed on a surface and whether they could adoptstructures that are recognized by proteins. Each of the 16 differentmolecular species occupies a different physical region on the glasssurface so that there is a one-to-one correspondence between molecularidentity and physical location. The molecules are of the formS-P-C-C-A/T-A/T-A/T-A/T-G-C-P-G-C-A/T-A/T-A/T-A/T-G-G-F where S is thesolid surface having silyl groups, P is a PEG linker, A, C, G, and T arethe DNA nucleotides, and F is a fluorescent tag. The DNA sequence islisted from the 3′ to the 5′ end (the 3′ end of the DNA molecule isattached to the solid surface via a silyl group and 2 PEG linkers). Thesixteen molecules synthesized on the solid support differed in thevarious permutations of A and T in the above formula.

Example 2

This example illustrates the ability of a library of surface-bound,unimolecular, double-stranded oligonucleotides to exist in duplex formand to be recognized and bound by a protein.

A library of 16 different members was prepared as described inExample 1. The 16 molecules all have the same composition (same numberof As, Cs, Gs and Ts), but the order is different. Four of the moleculeshave an outer strand that is 100% complementary to the inner strand(these molecules will be referred to as DS, double-stranded, below). Oneof the four DS oligonucleotides has a sequence that is recognized by therestriction enzyme EcoR1. If the molecule can loop back and form a DNAduplex, it should be recognized and cut by the restriction enzyme,thereby releasing the fluorescent tag. Thus, the action of the enzymeprovided a functional test for DNA structure, and also served todemonstrate that these structures can be recognized at the surface byproteins. The remaining 12 molecules had outer strands that were notcomplementary to their inner strands (referred to as SS,single-stranded, below). Of these, three had an outer strand and threehad an inner strand whose sequence was an EcoR1 half-site (the sequenceon one strand was correct for the enzyme, but the other half was not).The solid support with an array of molecules on the surface is referredto as a “chip” for the purposes of the following discussion. Thepresence of fluorescently labelled molecules on the chip was detectedusing confocal fluorescence microscopy. The action of various enzymeswas determined by monitoring the change in the amount of fluorescencefrom the molecules on the chip surface (e.g. “reading” the chip) upontreatment with enzymes that can cut the DNA and release the fluorescenttag at the 5′ end.

The three different enzymes used to characterize the structure of themolecules on the chip were:

-   1) Mung Bean Nuclease—sequence independent, single-strand specific    DNA endonuclease;-   2) DNase 1—sequence independent, double-strand specific    endonuclease;-   3) EcoR1—restriction endonuclease that recognizes the sequence    (5′-3′)

GAATTC in double stranded DNA, and cuts between the G and the first A.Mung Bean Nuclease and EcoR1 were obtained from New England Biolabs, andDNase I was obtained from Boehringer Mannheim. All enzymes were used ata concentration of 200 units per mL in the buffer recommended by themanufacturer. The enzymatic reactions were performed in a 1 mL flow cellat 22° C., and were typically allowed to proceed for 90 minutes.

Upon treatment of the chip with the enzyme EcoR1, the fluorescencesignal in the DS EcoR1 region and the 3 SS regions with the EcoR1half-site on the outer strand was reduced by about 10% of its initialvalue. This reduction was at least 5 times greater than for the otherregions of the chip, indicating that the action of the enzyme issequence specific on the chip. It was not possible to determine if thefactor is greater than 5 in these preliminary experiments because ofuncertainty in the constancy of the fluorescence background. However,because the purpose of these early experiments was to determine whetherunimolecular double-stranded structures could be formed and whether theycould be specifically recognized by proteins (and not to provide aquantitative measure of enzyme specificity), qualitative differencesbetween the different synthesis regions were sufficient.

The reduction in signal in the 3 SS regions with the EcoR1 half-site onthe outer strand indicated either that the enzyme cuts single-strandedDNA with a particular sequence, or that these molecules formed adouble-stranded structure that was recognized by the enzyme. Themolecules on the chip surface were at a relatively high density, with anaverage spacing of approximately 100 angstroms. Thus, it was possiblefor the outer strand of one molecule to form a double-stranded structurewith the outer strand of a neighboring molecule. In the case of the 3 SSregions with the EcoR1 half-site on the outer strand, such a bimoleculardouble-stranded region would have the correct sequence and structure tobe recognized by EcoR1. However, it would differ from the unimoleculardouble-stranded molecules in that the inner strand remainssingle-stranded and thus amenable to cleavage by a single-strandspecific endonuclease such as Mung Bean Nuclease. Therefore, it waspossible to distinguish unimolecular from bimolecular double-strandedDNA molecules on the surface by their ability to be cut by single anddouble-strand specific endonucleases.

In order to remove all molecules that have single-stranded structuresand to identify unimolecular double-stranded molecules, the chip wasfirst exhaustively treated with Mung Bean Nuclease. The reduction in thefluorescence signal was greater by about a factor of 2 for the SSregions of the chip, including those with the EcoR1 half-site on theouter strand that were cleaved by EcoR1, than for the 4 DS regions.Following Mung Bean Nuclease treatment, the chip was treated with eitherDNase I (which cuts all remaining double-stranded molecules) or EcoR1(which should cut only the remaining double-stranded molecules with thecorrect sequence). Upon treatment with DNase I, the fluorescence signalin the 4 DS regions was reduced by at least 5-fold more than the signalin the SS regions. Upon EcoR1 treatment, the signal in the single DSregion with the correct EcoR1 sequence was reduced by at least a factorof 3 more than the signal in any other region on the chip. Takentogether, these results indicated that the surface-bound moleculessynthesized with two complementary strands separated by a flexible PEGlinker form intramolecular double-stranded structures that wereresistant to a single-strand specific endonuclease and were recognizedby both a double-strand specific endonuclease, and a sequence-specificrestriction enzyme.

Example 3

This example illustrates the strategy employed for the preparation of aconformationally restricted hexapeptide.

A glass coverslip having aminopropylsilane spacer groups can be furtherderivatized on the amino groups with a poly-A oligonucleotide comprisingnine adenosine monomers using VLSIPS™ (“light-directed”) methods. Thetenth adenine monomer to be added will be a5′-aminopropyl-functionalized phosphoramidite (available from GlenResearch or Genosys Biotechnologies). To the amine terminus is thenadded, in stepwise fashion, the hexapeptide, RQFKVVT, beginning with thecarboxyl end of the peptide (i.e., as T-V-V-K-F-Q-R). A 3′-succinylatednucleoside can then be added under peptide coupling conditions and thenucleotide synthesis of the poly-T tail can be continued to provide aconformationally restricted probe.

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reviewing the above description. Thescope of the invention should, therefore, be determined not withreference to the above description, but should instead be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A synthetic unimolecular, double-stranded oligonucleotide librarycomprising a plurality of different members, each member having theformula:Y-L¹-X¹-L²-X² wherein, Y is a solid support: X¹ and X2 are a pair ofcomplementary oligonucleotides: L¹ is a spacer; L² is a linking grouphaving sufficient length such that X¹ and X² form a double-strandedoligonucleotide.
 2. A library in accordance with claim 1, wherein L² isa member selected from the group consisting of an alkylene group, apolyethyleneglycol group, a polyalcohol group, a polyamine group and apolyester group.
 3. A library in accordance with claim 1, wherein L² isa polyethylene glycol group.
 4. A library in accordance with claim 1,wherein X¹ and X² are complementary oligonucleotides each comprising offrom 6 to 30 nucleic acid monomers.
 5. A library in accordance withclaim 1, wherein said solid support is a silica support and L′ comprisesan aminoalkylsilane and from 1 to 4 hexaethyleneglycols.
 6. A library inaccordance with claim 1, wherein said solid support is a silica support,L¹ comprises an aminoalkylsilane and from 1 to 4 hexaethyleneglycols, L²is a polyethyleneglycol group and X¹ and X² are complementaryoligonucleotides each comprising of from 6 to 30 nucleic acid monomers.7. A synthetic unimolecular, double-stranded oligonucleotide library ofclaim 1, wherein a portion of said double-stranded oligonucleotidesformed by X¹ and X² further comprise a bulge.
 8. A syntheticunimolecular, double-stranded oligonucleotide library of claim 1,wherein a portion of said double-stranded oligonucleotides formed by X¹and X² further comprise a loop.
 9. A synthetic unimolecular,double-stranded nucleic acid library of claim 1, wherein each memberfurther comprises an identifier tag, said identifier tag identifying thesequence of said unimolecular, double-stranded nucleic acid.
 10. Asynthetic unimolecular, double-stranded nucleic acid library of claim 1,wherein said solid support comprises a first bead linked to a secondbead, wherein the double-stranded nucleic acid is attached to the firstbead and an identifier tag is attached to the second bead.
 11. A methodof forming a plurality of diverse unimolecular, double-strandedoligonucleotides on a solid support having optional spacers, saidsupport comprising a surface with a plurality of preselected regions,said method comprising: (a) forming on each of said preselected regionsa different first oligonucleotide, each of said first oligonucleotidescomprising of from 6 to 30 monomers; (b) attaching to the distal end ofeach of said first oligonucleotides of step (a) a linking group; and (c)forming on the distal end of each of said linking groups a secondoligonucleotide, wherein each of said second oligonucleotides iscomplementary to said first oligonucleotide which is attached within thesame preselected region, and wherein said linking groups have sufficientlength such that said first and second oligonucleotides form aunimolecular, double-stranded oligonucleotide.
 12. A method inaccordance with claim 11, wherein said method of construction of step(a) and step (b) is by light-directed synthesis. 13.-21. (canceled) 22.An adhesive for use in biological applications comprising a firstsurface having a plurality of attached oligonucleotides and a secondsurface having a plurality of attached oligonucleotides, wherein theoligonucleotides of said first surface are substantially complementaryto the oligonucleotides of said second surface.